T-cell receptor sequencing demonstrates persistence of virus-specific T cells after antiviral immunotherapy

Abstract

Viral infections are a serious cause of morbidity and mortality following haematopoietic stem cell transplantation (HSCT). Adoptive cellular therapy with virus-specific T cells (VSTs) has been successful in preventing or treating targeted viruses in prior studies, but the composition of ex vivo expanded VST and the critical cell populations that mediate antiviral activity in vivo are not well defined. We utilized deep sequencing of the T-cell receptor beta chain (TCRB) in order to classify and track VST populations in 12 patients who received VSTs following HSCT to prevent or treat viral infections. TCRB sequencing was performed on sorted VST products and patient peripheral blood mononuclear cells samples. TCRB diversity was gauged using the Shannon entropy index, and repertoire similarity determined using the Morisita-Horn index. Similarity indices reflected an early change in TCRB diversity in eight patients, and TCRB clonotypes corresponding to targeted viral epitopes expanded in eight patients. TCRB repertoire diversity increased in nine patients, and correlated with cytomegalovirus (CMV) viral load following VST infusion (P = 0·0071). These findings demonstrate that allogeneic VSTs can be tracked via TCRB sequencing, and suggests that T-cell receptor repertoire diversity may be critical for the control of CMV reactivation after HSCT.

Trial registration: ClinicalTrials.gov NCT00078533 NCT01945814.

Keywords: T-lymphocyte; adoptive immunotherapy; haematopoietic stem cell transplantation; viral infection.

Conflict of interest statement

Conflicts of Interests Disclosure: MDK, SD, HL, AR, CAL, YW, BJD, JH, RA, AJB, CR, DD have no relevant financial conflicts of interest to disclose. HEH is a founder of Viracyte and Marker Therapeutics and has received research support from Cell Medica and Tessa Therapeutics. CMB is a founder of Mana Therapeutics and received research support from Cellectis and NexImmune. PJH is a founder of Mana Therapeutics.

© 2019 British Society for Haematology and John Wiley & Sons Ltd.

Figures

Figure 1:. TRB gene segment usage demonstrates polyclonality of VST products.

A. T-cell receptor diversity of bulk virus-specific T cell (VST) products following 10-day ex vivo expansion demonstrates greater polyclonality (median Shannon entropy index 0.659), compared with Multimer-sorted VST samples corresponding to immunodominant cytomegalovirus (CMV) epitopes (median Shannon entropy index 0.413, *p=0.0009) or CD107A-sorted VST following CMV pepmix restimulation (median Shannon entropy index 0.419, **p=0.0105). B. Bulk VST products generally contained more unique T-cell receptor beta chain (TCRB) clonotypes (median 1477) in comparison to multimer sorted VST samples (median 97 clonotypes, not significant [ns]) or CD107A-sorted VST samples (median 124 clonotypes, ns). Lines/ranges: median/95% confidence interval. C.TRBV and TRBJ gene segment usage of the bulk product from Patient 8 (P8) versus multimer-sorted cells based on binding of a CMV-pp65/YSE pentamer. Colour coding denotes frequency of gene segment usage within the overall population.

Figure 2:. Virus-associated TCRB clonotypes expand longitudinally in the majority of responding patients.

Frequency of virus-associated T-cell receptor beta chain (TCRB) clonotypes in patient peripheral blood over time demonstrated expansion of clonotypes associated with cytomegalovirus/Epstein–Barr virus (CMV/EBV) epitopes in 8 of 11 evaluable patients within three months of virus-specific T cells (VST) infusion. Virus-associated clonotypes were detectable for up to four years post-infusion in Patient 1 (P1). Patient 5 (P5) had no antiviral response in spite of detectable expansion of clonotypes corresponding to the CMV-pp65/NLV epitope. Patient 10 (P10) had a high TCRB clonotype frequency corresponding to CMV-pp65/TPR prior to VST infusion, probably representing T cells from the maternal graft.

Figure 3:. CMV-pp65/NLV-specific T cells are detectable in a non-responding patient.

Multimer sorting of the virus-specific T cell (VST) product and peripheral blood in Patient 5 (P5) demonstrated the presence of cytomegalovirus (CMV)-specific CD8+ T cells recognizing the HLA-A02:01 restricted CMV-pp65/NLV epitope, which represented a third of CD8+ T cells in the VST product and 2.96% of CD8+ T cells in the peripheral blood of the patient at three weeks post-infusion.

Figure 4:. TCRB repertoires show similarity to CMV/EBV-associated clonotypes.

Similarity indices of the T-cell receptor beta chain (TCRB) repertoire comparing patient peripheral blood mononuclear cells with sorted or unsorted virus-specific T cell (VST) populations over time showed early repertoire changes in 8 of 11 evaluated patients. Five of nine responding patients (Patients 1, 2, 6, 9 and 11) displayed increasing similarity to the CMV/EBV pentamer-associated clonotypes after infusion, and Patient 8 had similarity to adenovirus pentamer-associated clonotypes at later timepoints. MH: Morisita-Horn Index.

Figure 5:. TCRB repertoire diversity increases in most responding patients and correlates with CMV control

. A. Normalized Shannon entropy index of the T-cell receptor beta chain (TCRB) repertoire increased over time in 9 of 12 evaluable patients, including 8 of 10 patients with complete responses. Patient 10 had a partial antiviral response to cytomegalovirus (CMV) with decreasing Shannon entropy index over time, and Patient 12 had a decrease in Shannon entropy index corresponding to corticosteroid treatment for GVHD. B. Shannon entropy index of high risk (CMV log>0, red) time points differed from low risk (CMV log 0, blue) time points in patients following virus-specific T cell (VST) infusion based on a logistical regression model (p=0.0071).